CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

Towards User-Centric Operation in 5G Networks

© 2016 Monserrat et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.There are three pillars that characterize the new 5G revolution, namely, the use of heterogeneous wireless access technologies conforming an ultra-dense network, the software-driven flexibility of this network, and the simplified and user-centric operation and management of the system. This next-generation network operation and management shall be based on the usage of Big Data Analytics techniques to monitor the end-user quality of experience through direct measures of the network. This paper describes the Astellia approach towards this network revolution and presents some results on the performance of quality estimation techniques in current cellular networks. Thanks to the use of this approach, operators may fill the gap of knowledge between network key performance indicators and user experience. This way, they can operate in a proactive manner and have actual measurements of the users' experience, which leads to a fairer judgement of the users' complaints.The authors would like to thank the funding received from the Ministerio de Industria, Energia y Turismo TSI-100102-2013-106 funds.Monserrat Del Río, JF.; Alepuz Benaches, I.; Cabrejas Peñuelas, J.; Osa Ginés, V.; López Bayo, J.; García-Zarza, R.; Domenech-Benlloch, MJ.... (2016). Towards User-Centric Operation in 5G Networks. EURASIP Journal on Wireless Communications and Networking. 2016(6):1-7. https://doi.org/10.1186/s13638-015-0506-zS1720166J Monserrat et al., Rethinking the mobile and wireless network architecture: the METIS research into 5G, in European Conference on Networks and Communications (EuCNC), 2014, pp. 1–55G-PPP, The 5G Infrastructure Public Private Partnership: the next generation of communication networks and services, 2015. Available at http://5g-ppp.eu/wp-content/uploads/2015/02/5G-Vision-Brochure-v1.pdfJF Monserrat, M Fallgren (eds.), Report on simulation results and evaluations, 2015. ICT-317669 METIS Deliverable 6.5Z Yingxiao, Z Ying Jun, User-centric virtual cell design for Cloud Radio Access Networks, in IEEE Signal Processing Advances in Wireless Communications (SPAWC), 2014, pp. 249–253JF Monserrat, G Mange, V Braun, H Tullberg, G Zimmermann, Ö Bulakci, METIS research advances towards the 5G mobile and wireless system definition. EURASIP. J. Wirel. Commun. Netw. 2015, 53 (2015)F Boccardi, RW Heath, A Lozano, TL Marzetta, P Popovski, Five disruptive technology directions for 5G. IEEE. Commun. Mag. 52(2), 74–80 (2014)P Agyapong, M Iwamura, D Staehle, W Kiess, A Benjebbour, Design considerations for a 5G network architecture. IEEE. Commun. Mag. 52(11), 65–75 (2014)Nokia Siemens Networks, Acquisition and retention white paper, 2013. http://networks.nokia.com/sites/default/files/document/acquisition___retention_white_paper.pdfDZ Yazti, S Krishnaswamy, Mobile big data analytics: research, practice, and opportunities, in IEEE 15th International Conference on Mobile Data Management (MDM), 2014R Kreher, UMTS performance measurement: a practical guide to KPIs for the UTRAN environment (Wiley, Chichester, 2006)S Mehrotra, On the implementation of a primal-dual interior point method. SIAM. J. Optim. 2, 575–601 (1992)V Osa, J Matamales, J Monserrat, J Lopez, Localization in wireless networks: the potential of triangulation techniques. Wirel. Pers. Commun. 68(4), 1525–1538 (2013

Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins

Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction

Visualizing Spatiotemporal Dynamics of Intercellular Mechanotransmission upon Wounding.

During cell-to-cell communications, the interplay between physical and biochemical cues is essential for informational exchange and functional coordination, especially in multicellular organisms. However, it remains a challenge to visualize intercellular signaling dynamics in single live cells. Here, we report a photonic approach, based on laser microscissors and Förster resonance energy transfer (FRET) microscopy, to study intercellular signaling transmission. First, using our high-throughput screening platform, we developed a highly sensitive FRET-based biosensor (SCAGE) for Src kinase, a key regulator of intercellular interactions and signaling cascades. Notably, SCAGE showed a more than 40-fold sensitivity enhancement than the original biosensor in live mammalian cells. Next, upon local severance of physical intercellular connections by femtosecond laser pulses, SCAGE enabled the visualization of a transient Src activation across neighboring cells. Lastly, we found that this observed transient Src activation following the loss of cell-cell contacts depends on the passive structural support of cytoskeleton but not on the active actomyosin contractility. Hence, by precisely introducing local physical perturbations and directly visualizing spatiotemporal transmission of ensuing signaling events, our integrated approach could be broadly applied to mimic and investigate the wounding process at single-cell resolutions. This integrated approach with highly sensitive FRET-based biosensors provides a unique system to advance our in-depth understanding of molecular mechanisms underlying the physical-biochemical basis of intercellular coupling and wounding processes

Visualizing the mechanical activation of Src

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations